This protocol is designed to passage stem cell colonies cultured in a 6 cm dish, using Stem Cell Dissociation Reagent (ATCC ACS-3010) to detach the cell colonies. Stem Cell Dissociation Reagent is stored as a 0.5 U/mL working solution at -20°C. The cells should be split at a ratio of 1:4.
Volumes should be adjusted according to the size and number of the tissue culture vessels to be processed.
1. Warm an aliquot of Stem Cell Dissociation Reagent working solution to room temperature.
2. Aspirate and discard the stem cell culture medium.
3. Rinse the cells twice with 4 mL of D-PBS (ATCC 30-2200).
4. Add 2 mL of Stem Cell Dissociation Reagent working solution to the dish.
5. Incubate at 37°C for 10 to 15 minutes or until the edges of the individual colonies begin to loosen and fold back. View the dish under the microscope starting at 5 minutes as incubation time will vary depending on the cell line and colony size.
6. Aspirate the Stem Cell Dissociation Reagent and gently rinse the colonies with 4 mL of DMEM: F-12 Medium (ATCC 30-2006), taking care not to dislodge the cells during manipulation.
7. Add 2 mL of Pluripotent Stem Cell SFM XF/FF to the dish, and detach the cells by pipetting up and down several times with a 1 mL tip. Take care not to over-pipette the culture into a single-cell suspension as single cells will not establish colonies after seeding.
8. Transfer the cell aggregates to a 15 mL conical tube.
9. Add an additional 3 mL of Pluripotent Stem Cell SFM XF/FF to the dish to collect any remaining cells. Transfer this rinse to the 15 mL conical tube containing the cell aggregates.
10. Centrifuge the cell aggregates at 200 x g for 5 minutes.
11. Aspirate the supernatant and discard.
12. Gently resuspend the pellet by pipetting up and down 5 to 6 times with a 1 mL tip, maintaining the small cell aggregates.
13. Plate the cells as desired on feeder or feeder-free cultures in the presence of 10 µM ROCK Inhibitor Y27632* (ATCC ACS-3030) to the cell culture medium is recommended.
*ROCK Inhibitor Y27632 has been shown to reduce dissociation-induced apoptosis.
14. Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day. Passage the cells every 4 to 5 days (80% confluent).
For optimal results, cryopreserve stem cell colonies when the cell cultures are 80% confluent. This protocol is designed to cryopreserve stem cell colonies cultured in a 6 cm dish.
- Detach stem cell colonies from the dish as described in the recommended subculturing protocol (steps 1-11). Gently tap the bottom of the tube to loosen the cell pellet.
- Take the Stem Cell Freezing Media from storage and swirl to mix. Keep cold. Decontaminate by dipping in or spraying with 70% alcohol.
- Add 2 mL of cold Stem Cell Freezing Media to the tube. Gently resuspend the pellet by pipetting up and down 5-6 times with a 1 mL tip, maintaining the cell aggregates.
- Immediately transfer 1 mL each of the cell suspension into two labeled cryovials.
- Freeze the cells gradually at a rate of -1°C/min until the temperature reaches -70°C to -80°C. An isopropanol freezing container also may be used.
- The cells should not be left at -80°C for more than 24 to 48 hours. Once at -80°C, frozen cryovials should be transferred to the vapor phase of liquid nitrogen for long-term storage.
Pluripotent Stem Cell SFM XF/FF, ATCC ACS-3002
CellMatrix Basement Membrane Gel, ATCC ACS-3035